Degenerate DNA recognition by I-PpoI endonuclease.

The I-PpoI endonuclease is encoded by a group I intron found in the slime mold Physarum polycephalum. To initiate homing of its encoding intron, I-PpoI catalyzes a specific double-stranded break within a 15-bp recognition site. The high substrate specificities of I-PpoI and other homing endonucleases make these enzymes valuable tools for genomic mapping and sequencing. Here, we report on the ability of I-PpoI to cleave recognition sites that contain a wide variety of mutations generated randomly or deliberately. We find that much degeneracy is tolerated within the recognition site of I-PpoI. Few single substitutions prevent cleavage completely. In addition, many sites with multiple substitutions are cleaved efficiently. In contrast, deletions or insertions within the I-PpoI recognition site are detrimental to catalysis, indicating that proper registry between the protein and its substrate is critical. Finally, we find that the sequence of the flanking regions can influence catalysis by I-PpoI. Thus, I-PpoI has both the complex binding specificity of a transcription factor and the catalytic ability of a restriction endonuclease.

Contribution of a tyrosine side chain to ribonuclease A catalysis and stability.

An intricate architecture of covalent bonds and noncovalent interactions appear to position the side chain of Lys 41 properly within the active site of bovine pancreatic ribonuclease A (RNase A). One of these interactions arises from Tyr 97, which is conserved in all 41 RNase A homologues of known sequence. Tyr 97 has a solvent-inaccessible side chain that donates a hydrogen bond to the main-chain oxygen of Lys 41. Here, the role of Tyr 97 was examined by replacing Tyr 97 with a phenylalanine, alanine, or glycine residue. All three mutant proteins have diminished catalytic activity, with the value of Kcat being perturbed more significantly than that of Km. The free energies with which Y97F, Y97A, and Y97G RNase A bind to the rate-limiting transition state during the cleavage of poly(cytidylic acid) are diminished by 0.74, 3.3, and 3.8 kcal/mol, respectively. These results show that even though Tyr 97 is remote from the active site, its side chain contributes to catalysis. The role of Tyr 97 in the thermal stability of RNase A is large. The conformational free energies of native Y97F, Y97A, and Y97G RNase A are decreased by 3.54, 12.0, and 11.7 kcal/mol, respectively. The unusually large decrease in stability caused by the Tyr-->Phe mutation could result from a decrease in the barrier to isomerization of the Lys 41-Pro 42 peptide bond.

Substrate binding and turnover by the highly specific I-PpoI endonuclease.

Intron-encoded endonucleases are distinguished by their ability to catalyze the cleavage of double-stranded DNA with high specificity. I-PpoI endonuclease, an intron-encoded endonuclease from the slime mold Physarum polycephalum, is a small enzyme (2 x 20 kDa) that catalyzes the cleavage of a large asymmetric DNA sequence (15 base pairs). Here, the interactions of I-PpoI with its substrate were examined during both binding (in the absence of Mg2+) and catalysis (in the presence of Mg2+). Using circular permutation assays, I-PpoI was shown to bend its substrate by 38 +/- 4 degrees upon binding. Two independent methods, gel mobility shift assays and fluorescence polarization assays, revealed that I-PpoI binds tightly to its substrate. Values of Kd range from 3.3 to 112 nM, increasing with increasing NaCl concentration. Similar salt effects on the values of Km were observed during steady-state catalysis. At low salt concentrations, the value of kcat/Km for the cleavage of an oligonucleotide duplex approaches 10(8) M-1 s-1. Although other divalent cations can replace Mg2+, catalysis by I-PpoI is most efficient in the presence of an oxophilic metal ion that prefers an octahedral geometry: Mg2+ > Mn2+ > Ca2+ = Co2+ > Ni2+ > Zn2+. Together, these results provide the first chemical insight into substrate binding and turnover by an intron-encoded endonuclease.